(1) Field of the Invention
The present invention relates to plant cells and plants which synthesize hyaluronan, and to methods for preparing such plants, and also to methods for preparing hyaluronan with the aid of these plant cells or plants. Furthermore, the present invention relates to the use of plants for preparing hyaluronan and hyaluronan-containing food or feed.
(2) Description of the Related Art
Hyaluronan is a naturally occurring unbranched, linear mucopolysaccharide (glucosaminoglucan) which is constructed of alternating molecules of glucuronic acid and. N-acetyl-glucosamine. The basic building block of hyaluronan consists of the disaccharide glucuronic acid-beta-1,3-N-acetyl-glucosamine. In hyaluronan, these repeating units are attached to one another by beta-1,4 linkages.
In pharmacy, use is frequently made of the term hyaluronic acid. Since hyaluronan is in most cases present as polyanion and not as free acid, hereinbelow, the term hyaluronan is preferably used, but each term is to be understood as embracing both molecular forms.
Hyaluronan has unusual physical chemical properties, such as, for example, properties of polyelectrolytes, viscoelastic properties, a high capacity to bind water, properties of gel formation, which, in addition to further properties of hyaluronan, are described in a review article by Lapcik et al. (1998, Chemical Reviews 98(8), 2663-2684). The specific properties of hyaluronan are determined inter alia by the molecular weight and the molecular weight distribution of the hyaluronan in question.
Hyaluronan is a component of extracellular connective tissue and bodily fluids of vertebrates. In humans, hyaluronic acid is synthesized by the cell membrane of all body cells, especially mesenchymal cells, and ubiquitously present in the body with a particularly high concentration in the connective tissues, the extracellular matrix, the umbilical cord, the joint fluid, the cartilageous tissue, the skin and the vitreous body of the eye (Bernhard Gebauer, 1998, Inaugural-Dissertation, Virchow-Klinikum Medizinische Fakultät Charité der Humboldt Universität zu Berlin; Fraser et al., 1997, Journal of Internal Medicine 242, 27-33).
Recently, hyaluronan was also found in animal non-vertebrate organisms (molluscs) (Volpi and Maccari, 2003, Biochimie 85, 619-625).
Furthermore, some pathogenic gram-positive bacteria (Streptococcus group A and C) and gram-negative bacteria (Pasteurella) synthesize hyaluronan as exopolysaccharides which protect these bacteria against attack by the immune system of their host, since hyaluronan is a non-immunogenic substance.
Viruses which infect single-cell green algae of the genus Chlorella, some of which are present as endosymbionts in Paramecium species, bestow upon the single-cell green algae the ability to synthesize hyaluronan after infection by the virus (Graves et al., 1999, Virology 257, 15-23). Hitherto, this is the only example from the systematic kingdom of the plants where the synthesis of hyaluronan was demonstrated. However, the ability to synthesize hyaluronan is not a feature which characterizes the algae in question. The ability of the algae to synthesize hyaluronan is mediated by an infection with a virus whose genome has a sequence coding for hyaluronan synthase (DeAngelis, 1997, Science 278, 1800-1803). Furthermore, the virus genome contains sequences coding for a UDP-glucose dehydrogenase (UDP-Glc-DH) and a glutamine: fructose 6-phosphate amidotransferase (GFTA). UDP-Glc-DH catalyses the synthesis of UDP-glucuronic acid used as substrate by hyaluronan synthase. GFTA converts fructose 6-phosphate into glucosamine 6-phosphate which is an important metabolite in the metabolic pathway for hyaluronan synthesis. Both genes encode active proteins which, like the hyaluronan synthase of the virus, are transcribed simultaneously in the early phase of the viral infection (DeAngelis et al., 1997, Science 278, 1800-1803, Graves et al., 1999, Virology 257, 15-23). Plants themselves do not have any nucleic acids in their genome which code for proteins catalysing the synthesis of hyaluronan and, although a large number of plant carbohydrates have been described and characterized, it has hitherto not been possible to detect hyaluronan or molecules related to hyaluronan in non-infected plants (Graves et al., 1999, Virology 257, 15-23).
The catalysis of the hyaluronan synthesis is effected by a single membrane-integrated or membrane-associated enzyme, hyaluronan synthase. The hyaluronan synthases which have hitherto been studied can be classified into two groups: hyaluronan synthases of Class I and hyaluronan synthases of Class II (DeAngelis, 1999, CMLS, Cellular and Molecular Life Sciences 56, 670-682).
The hyaluronan synthases of vertebrates are further distinguished by the identified isoenzymes. The different isoenzymes are referred to in the order of their identification using Arabic numbers (for example, hsHAS1, hsHAS2, hsHAS3).
The unusual properties of hyaluronan offer a wealth of possibilities for application in various fields, such as, for example, pharmacy, the cosmetics industry, in the production of food and feed, in technical applications (for example as lubricants), etc. The most important applications where hyaluronan is currently being used are in the medicinal and cosmetics field (see, for example, Lapcik et al., 1998, Chemical Reviews 98(8), 2663-2684, Goa and Benfield, 1994, Drugs 47(3), 536-566).
In the medical field, hyaluronan-containing products are currently used for the intraarticular treatment of arthrosis and in ophthalmics used for eye surgery. Hyaluronan is also used for treating joint disorders in racehorses. In addition, hyaluronic acid is a component of some rhinologics which, for example in the form of eye drops and nasalia, serve to moisten dry mucous membranes. Hyaluronan-containing solutions for injection are used as analgesics and antirheumatics. Patches comprising hyaluronan or derivatized hyaluronan are employed in wound healing. As dermatics, hyaluronan-containing gel implants are used for correcting skin deformations in plastic surgery.
For pharmacological applications, preference is given to using hyaluronan having a high molecular weight.
In cosmetic medicine, hyaluronan preparations are among the most suitable skin filler materials. By injecting hyaluronan, for a limited period of time, it is possible to smooth wrinkles or to increase the volume of lips.
In cosmetic products, in particular in skin creams and lotions, hyaluronan is frequently used as moisturizer by virtue of its high water-binding capacity.
Further possibilities of application in the medicinal and cosmetics field, such as, for example, the use of hyaluronan as carrier for active compounds which ensures a controlled release of the active compound over a long period of time, as carrier for active compounds which transports the active compounds in a targeted manner into the lymphatic system or as active compound which, after application as an ointment, ensures that the active compound remains in the skin for a relatively long period of time, are described in Lapcik et al. (1998, Chemical Reviews 98(8), 2663-2684). The use of hyaluronan derivatives in the medicinal field requires further research efforts; however, first results have already revealed a large potential (Lapcik et al. 1998, Chemical Reviews 98(8), 2663-2684).
Furthermore, hyaluronan-containing preparations are sold as so-called nutraceuticals (food supplements) which can also be used in animals (for example dogs, horses) for the prophylaxis and alleviation of arthrosis.
Hyaluronan used for commercial purposes is currently isolated from animal tissues (rooster combs) or prepared fermentatively using bacterial cultures.
U.S. Pat. No. 4,141,973 describes a process for isolating hyaluronan from rooster combs or alternatively from umbilical cords. In addition to hyaluronan, animal tissues (for example rooster combs, umbilical cords) also contain further mucopolysaccharides related to hyaluronan, such as chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate and heparin. Furthermore, animal organisms contain proteins (hyaladherins) which bind specifically to hyaluronan and which are required for the most different functions in the organism, such as, for example, the degradation of hyaluronan in the liver, the function of hyaluronan as lead structure for cell migration, the regulation of endocytosis, the anchoring of hyaluronan on the cell surface or the formation of hyaluronan networks (Turley, 1991, Adv Drug Delivery Rev 7, 257 ff.; Laurent and Fraser, 1992, FASEB J. 6, 183 ff.; Stamenkovic and Aruffo, 1993, Methods Enzymol. 245, 195 ff; Knudson and Knudson, 1993, FASEB 7, 1233 ff.).
The Streptococcus strains used for the bacterial production of hyaluronan are exclusively pathogenic bacteria. During cultivation, too, these bacteria produce (pyrogenic) exotoxins and haemolysins (streptolysin, (in particular alpha- and beta-haemolysin) (Kilian, M.: Streptococcus and Enterococcus. In: Medical Microbiology. Greenwood, D.; Slack, R C A; Peutherer, J. F. (Eds.). Chapter 16. Churchill Livingstone, Edinburgh, UK: pp. 174-188, 2002, ISBN 0443070776) which are released into the culture medium. This renders purification and isolation of the hyaluronan prepared with the aid of Streptococcus strains more difficult. In particular for pharmaceutical application, the presence of exotoxins and haemolysins in the preparation is a problem.
U.S. Pat. No. 4,801,539 describes the preparation of hyaluronan by fermentation of a mutagenized bacterial strain (Streptococcus zooedemicus). The mutagenized bacteria strain used no longer synthesizes beta-haemolysin. The yield achieved was 3.6 g of hyaluronan per liter of culture.
EP 0694616 describes a method for cultivating Streptococcus zooedemicus or Streptococcus equi, where, under the culture conditions employed, no streptolysin, but increased amounts of hyaluronan are synthesized. The yield achieved was 3.5 g of hyaluronan per liter of culture.
During cultivation, Streptococcus strains release the enzyme hyaluronidase into the culture medium, as a consequence of which, in this production system, too, the molecular weight is reduced during purification. The use of hyaluronidase-negative Streptococcus strains or of methods for the production of hyaluronan where the production of hyaluronidase during cultivation is inhibited are described in U.S. Pat. No. 4,782,046. The yield achieved was up to 2.5 g of hyaluronan per liter of culture, and the maximum mean molecular weight achieved was 3.8×106 Da, at a molecular weight distribution of from 2.4×106 to 4.0×106.
US 20030175902 and WO 03 054163 describe the preparation of hyaluronan with the aid of heterologous expression of a hyaluronan synthase from Streptococcus equisimilis in Bacillus subtilis. To achieve the production of sufficient amounts of hyaluronan, in addition to heterologous expression of a hyaluronan synthase, simultaneous expression of an UDP-glucose dehydrogenase in the Bacillus cells is also required. US 20030175902 and WO 03 054163 do not state the absolute amount of hyaluronan obtained in the production with the aid of Bacillus subtilis. The maximum mean molecular weight achieved was about 4.2×106. However, this mean molecular weight was only achieved for the recombinant Bacillus. strain where a gene coding for the hyaluronan synthase gene from Streptococcus equisimilis and the gene coding for the UDP-glucose dehydrogenase from Bacillus subtilis were integrated into the Bacillus subtilis genome under the control of the amyQ promoter, where at the same time the Bacillus subtilis-endogenous cxpY gene (which codes for a cytochrome P450 oxidase) was inactivated.
The production of hyaluronan by fermentation of bacteria strains is associated with high costs, since the bacteria have to be fermented in sealed sterile containers under expensive controlled culture conditions (see, for example, U.S. Pat. No. 4,897,349). Furthermore, the amount of hyaluronan which can be produced by fermentation of bacteria strains is limited by the production facilities present in each case. Here, it also has to be taken into account that fermenters, as a consequence of physical laws, cannot be built for excessively large culture volumes. Particular mention may be made here of homogeneous mixing of the substances fed in from the outside (for example essential nutrient sources for bacteria, reagents for regulating the pH, oxygen) with the culture medium required for efficient production, which, in large fermenters, can be ensured only with great technical expenditure, if at all.
The purification of hyaluronan from animal organisms is complicated owing to the presence, in animal tissues, of other mucopolysaccharides and proteins which specifically bind to hyaluronan. In patients, the use of hyaluronan-containing medicinal preparations contaminated by animal proteins can result in unwanted immunological reactions of the body (U.S. Pat. No. 4,141,973), in particular if the patient is allergic to animal proteins (for example chicken egg white). Furthermore, the amounts (yields) of hyaluronan which can be obtained from animal tissues in satisfactory quality and purity are low (rooster comb: 0.079% w/w, EP 0144019, U.S. Pat. No. 4,782,046), which necessitates the processing of large amounts of animal tissues. A further problem in the isolation of hyaluronan from animal tissues consists in effect that the molecular weight of hyaluronan during purification is reduced since animal tissues also contain a hyaluronan-degrading enzyme (hyaluronidase).
In addition to the hyaluronidases and exotoxins mentioned, Streptococcus strains also produce endotoxins which, when present in pharmacological products, pose risks for the health of the patient. In a scientific study, it was shown that even hyaluronan-containing medicinal products on the market contain detectable amounts of bacterial endotoxins (Dick et al., 2003, Eur J Opthalmol. 13(2), 176-184). A further disadvantage of the hyaluronan produced with the aid of Streptococcus strains is the fact that the isolated hyaluronan has a lower molecular weight than hyaluronan isolated from rooster combs (Lapcik et al. 1998, Chemical Reviews 98(8), 2663-2684). US 20030134393 describes the use of a Streptococcus strain for producing hyaluronan which synthesizes a particularly pronounced hyaluronan capsule (supercapsulated). The hyaluronan isolated after fermentation had a molecular weight of 9.1×106. However, the yield was only 350 mg per liter.
Although hyaluronan has unusual properties, it is, owing to its scarcity and the high price, rarely, if at all, used for industrial applications.